Heredity (2016) 116, 304–313
& 2016 Macmillan Publishers Limited All rights reserved 0018-067X/16
www.nature.com/hdy
ORIGINAL ARTICLE
Reduction in the cumulative effect of stress-induced
inbreeding depression due to intragenerational purging
in Drosophila melanogaster
LS Enders1,2 and L Nunney1
Environmental stress generally exacerbates the harmful effects of inbreeding and it has been proposed that this could be
exploited in purging deleterious alleles from threatened inbred populations. However, understanding what factors contribute to
variability in the strength of inbreeding depression (ID) observed across adverse environmental conditions remains a challenge.
Here, we examined how the nature and timing of stress affects ID and the potential for purging using inbred and outbred
Drosophila melanogaster larvae exposed to biotic (larval competition, bacteria infection) and abiotic (ethanol, heat) stressors
compared with unstressed controls. ID was measured during (larval survival) and after (male mating success) stress exposure.
The level of stress imposed by each stressor was approximately equal, averaging a 42% reduction in outbred larval survival
relative to controls. All stressors induced on average the same ID, causing a threefold increase in lethal equivalents for larval
survival relative to controls. However, stress-induced ID in larval success was followed by a 30% reduction in ID in mating
success of surviving males. We propose that this fitness recovery is due to ‘intragenerational purging’ whereby fitness correlations
facilitate stress-induced purging that increases the average fitness of survivors in later life history stages. For biotic stressors,
post-stress reductions in ID are consistent with intragenerational purging, whereas for abiotic stressors, there appeared to be an
interaction between purging and stress-induced physiological damage. For all stressors, there was no net effect of stress on
lifetime ID compared with unstressed controls, undermining the prediction that stress enhances the effectiveness of populationlevel purging across generations.
Heredity (2016) 116, 304–313; doi:10.1038/hdy.2015.103; published online 25 November 2015
INTRODUCTION
Wild populations face a continuous onslaught of natural and anthropogenic stresses including seasonal changes, drought, disease, pollution,
habitat loss and competition from invasive species (Loeschcke et al.,
2004; Frankham, 2005). When taken to extremes, environmental stress
can threaten the survival of a population, but more generally the effect
of stress is to significantly reduce the fitness of individuals in a
population relative to more benign conditions (Hoffmann and
Parsons, 1991). As a result, stress is recognized as having major
implications for both short-term survival and long-term adaptation
(Loeschcke et al., 2004; Frankham, 2005; Bijlsma and Loeschcke, 2012).
The deleterious effects of stress are often amplified in inbred
individuals (Armbruster and Reed, 2005; Fox and Reed, 2011),
rendering small inbred populations particularly vulnerable to stressful
conditions (Reed et al., 2002; Bijlsma and Loeschcke, 2012).
Individuals may be inbred because their parents are close relatives
or because they are members of a small population where all
individuals share a high degree of coancestry. In either case, increased
inbreeding increases the probability that two gene copies chosen at
random are identical by descent, measured by the inbreeding
coefficient (F) (Wright, 1931). The result is often inbreeding depression (ID), a reduction in fitness primarily due to the expression of
recessive deleterious alleles (Charlesworth and Willis, 2009). Relative
to benign conditions, environmental stress is predicted to exacerbate
ID by increasing the number of alleles with deleterious effects and/or
by amplifying the effects of deleterious alleles already expressed under
benign conditions (Bijlsma et al., 1999; Reed et al., 2012). However,
periods of stress may reverse the buildup of ID through a process of
genetic purging. Purging is a reduction in the frequency of deleterious
recessive alleles owing to their increased exposure to selection under
inbreeding (Hedrick, 1994; Wang, 2000) and has been proposed as a
potential tool for managing ID in endangered and captive populations
(Swindell and Bouzat, 2006; de Cara et al., 2013). The effectiveness of
purging in a population is predicted to increase during exposure to
stress, given that stress can magnify the strength of selection against
deleterious alleles (Bijlsma et al., 1999; Plough, 2012; Reed et al., 2012).
Although ID is a common phenomenon in nature, understanding
what factors contribute to the high degree of variability in magnitude
observed across environments remains a challenge (Armbruster and
Reed, 2005; Fox and Reed, 2011; Yun and Agrawal, 2014). Predicting
the outcome of inbreeding-stress interactions centers on the concept
that stress alters the strength of selection against deleterious alleles that
cause ID (Agrawal and Whitlock, 2010; Yun and Agrawal, 2014).
The finding that the intensity of stress scales positively with ID in a
variety of taxa (Fox and Reed, 2011; Enders and Nunney, 2012; Schou
et al., 2015) supports the general prediction that selection is greater in
1
Department of Biology, University of California, Riverside, Riverside, CA, USA and 2Department of Entomology, University of Nebraska-Lincoln, Lincoln, NE, USA
Correspondence: Dr LS Enders, Department of Entomology, University of Nebraska-Lincoln, 103 Entomology Hall, Lincoln, NE 68583, USA.
E-mail: [email protected]
Received 16 June 2015; revised 5 October 2015; accepted 6 October 2015; published online 25 November 2015
Stress and purging reduce inbreeding depression
LS Enders and L Nunney
305
stressful environments. However, different stress types have been
shown to vary in their ability to increase selection against mutations
(Agrawal and Whitlock, 2010). Recent work in Drosophila also found
the degree of density-dependent intraspecific competition associated
with a stressful environment was a better predictor of ID than the level
of stress alone (Yun and Agrawal, 2014), further suggesting that
stressor-specific factors influence the expression of ID.
Environmental stress can be broadly categorized as abiotic or biotic,
and within these two groups, there exists a wide range of stress types.
Abiotic examples include extreme temperatures, chemicals and
desiccation, whereas biotic examples include the presence of predators
or parasites, infection with pathogens and intense competition.
Organisms often exhibit varied responses to different forms of stress
(Kültz, 2005; Atkinson and Urwin, 2012), and plants show characteristic differences in their response to biotic vs abiotic stressors
(see Atkinson and Urwin, 2012), suggesting that stressor-specific
interactions with the expression of deleterious alleles (that is, the
genetic load) may contribute to variation in the expression of ID.
Fundamental differences in the way in which unique stressors affect
the expression of genes and hence the genetic load could translate into
qualitatively different outcomes for the survival of inbred and outbred
individuals (Cheptou and Donohue, 2011; Reed et al., 2012).
In particular, the potential for biotic stressors to alter the strength
and nature of stress imposed on other organisms suggests that their
interactions with inbreeding could differ relative to abiotic forms of
stress. However, detection of stress-specific effects on ID is difficult
unless the level of stress imposed by different stressors is somehow
standardized. Ideally, comparison of ID across stress types should be
performed using equal stress levels, although regression-based corrections for the variation in the stress level may also be justifiable.
Given the view that stress increases ID through its effects on the
expression of deleterious alleles, it is important to know whether these
effects cause permanent developmental changes or have a transient
effect. The prevailing view is that environmental stress causes general
and persistent physiological weakening, the effect of which is greater in
inbred individuals. This hypothesis predicts that stress imposed early
in life (for example, during development) will lead to increased ID
across fitness traits expressed later in life (for example, mating success,
fecundity), including periods following direct exposure to stress when
conditions are benign (Hoffmann and Parsons, 1991; Armbruster and
Reed, 2005; Waller et al., 2008). However, an alternative hypothesis
predicts the opposite trend in ID following exposure to stress early in
life. This hypothesis invokes intragenerational purging, whereby a
period of stress during early life history stages selects against
deleterious homozygous genotypes, leading to an increase in the
average fitness of the surviving population and subsequent reduction
in ID in later life (Enders and Nunney, 2010; Goodrich et al., 2013).
Intragenerational stress-induced purging requires that fitness correlations exist across multiple life history stages, such that deleterious
mutations affecting early developmental stages also adversely affect
adult fitness components, such as mating success or fecundity
(Goodrich et al., 2013). These two hypotheses are not exclusive
because stress-induced purging and physiological weakening may
function simultaneously, with their counteracting effects resulting in
little or no change in levels of ID across life history stages or
cumulatively over the life span of an organism.
There is some experimental support for the view that short-term
exposure to stress at one stage can reduce (or at least not increase) ID
at a later developmental stage in plants (Montalvo, 1994; Goodrich
et al., 2013) and Drosophila (Enders and Nunney, 2010). In general, it
is unknown which (or whether all) types of stress lower ID in later life
history stages. Specific stressors can increase, decrease or have no
effect on the magnitude of selection against mutations (Agrawal and
Whitlock, 2010), and the resulting variation in the effect of different
stressors could contribute to differences in the degree of intragenerational purging across stress types (Kristensen et al., 2003;
Swindell and Bouzat, 2006). Likewise, differences in the extent to
which unique stressors inflict irreversible cellular damage could
produce varying degrees of physiological weakening that in turn
influence the expression of ID and could counteract the fitness effects
of intragenerational purging.
The aim of the present work was to investigate two unresolved
aspects of the effect of stress on ID. First, the extent to which different
stress types lead to different levels of ID. Second, the extent to which
stress imposed early in the life of an organism affects the level of ID
expressed later in life, specifically whether ID is increased
(the physiological weakening hypothesis) or decreased (the intragenerational purging hypothesis). We investigated these two issues in
D. melanogaster by applying four types of environmental stress during
the larval stage across three levels of inbreeding (F = 0, 0.25 and 0.50).
We measured ID expressed for larval survival (LS) (reflecting the
direct effects of the stress) and for male mating success (post stress).
We used male mating success as an indicator of post-stress performance because we had previously shown that the effects of sexual
selection exacerbated the expression of ID, so that the effect on males
was much greater than that on females (Enders and Nunney, 2010).
To compare the effect of the four different kinds of stress, an
important feature of the experimental design was that we imposed
approximately equal levels of stress with all of the stressors, where the
level of stress was defined as the drop in LS observed in the stressed
outbred population.
MATERIALS AND METHODS
Base population and inbreeding design
In October 2008, outbred stock populations of D. melanogaster were collected
from two locations in Northern California, the Galante Winery in Carmel
Valley (36°24’4.827’’ N, 121°39’26.476’’ W) (Gala) and the Mayo Family
Winery in Sonoma Valley (38°22’13.868’’ N, 122°21’0.13’’ W) (Mayo).
To minimize any modification of the genetic architecture through selection
or inbreeding, 400 pairs of wild caught flies from each location were placed in
vials and reared in the laboratory at 18 °C. Their progeny were outcrossed by
taking a single male and female from each of the 400 pairs (per population) and
mating them in a circular design, whereby each male is mated to the female
from the next vial. Each of the two outbred stock populations (Gala and Mayo)
were reared at 18 °C using the above circular outcrossing design for six
generations prior to the start of the experiment. As each pair contributed
equally to the next generation, laboratory selection was minimized because it
was restricted to within-family effects, and because the male and female of each
pair was unrelated, inbreeding was also minimized.
From both of the large outbred stock populations described above, 10 inbred
lines were created for each of two levels of inbreeding (F = 0.25, 0.5) using the
appropriate number of generations of full-sib mating. To create each of these
inbred lines (2 source populations × 2 levels of F × 10 replicate lines), five
replicate full-sib pairs were set up in separate vials each generation.
Ten offspring of one of the five pairs, chosen at random, were used to establish
the next generation of sibling mating (five new sibling pairs). This method of
sibling mating was carried out for three generations to reach an F = 0.50 and for
one generation to reach an F = 0.25. The level of inbreeding (F) was calculated
according to Wright’s equation for full-sib mating: Ft = (1+ 2Ft-1–Ft-2)/4
(Falconer and Mackay, 1996). Both of the two large outbred stock populations
(F = 0) were maintained simultaneously as 400 pairs of individuals, using the
circular mating design described above. All of the above inbred and outbred
lines were reared at 25 °C.
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Stress and purging reduce inbreeding depression
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All inbred lines and the outbred base population were synchronized so that
individuals reached the desired level of inbreeding (F = 0, 0.25 or 0.50) at the
same time. When the inbred lines had reached the desired level of F, they were
expanded to large inbred bottle populations (N = 100) to rear enough adults for
use in the experiment. This was carried out by taking 100 virgin progeny
(50 males: 50 females) from a single sib pair at the final generation of sib
mating and placing them into a large bottle to randomly mate. After 1 week, the
adults were transferred to a second bottle of new food. The progeny of these
two bottles were then used to create two replicate experimental bottle
populations (N = 100, 50:50 virgin males and females) for each of the 20
inbred lines (10 each at F = 0.25 and 0.50) per source population. Five replicate
outbred lines (bottle populations with N = 100) were created from each of the
two source populations by combining one virgin male and female from each of
50 randomly chosen pairs from the 400 stock pairs used to maintain the
outbred populations. The offspring (F1) of the inbred and outbred bottle
populations were used in the experimental treatments described below. The
experiment was replicated twice (Block I and II) with all lines from both source
populations (Gala and Mayo).
Environmental stress treatments
In all of the 100 tests (2 blocks × (5 lines at F = 0; 10 at F = 0.25; 10 at
F = 0.5) × 2 source populations), the F1 larvae were subject to four types of
stress plus control conditions in vials containing 10 ml of the appropriate food
medium. Each condition was replicated three times per test. The control vials
contained a standard food medium consisting of molasses, cornmeal, yeast,
water and the antifungal agent Tegosept. The abiotic stress treatments were:
(i) heat stress, where larvae were reared under fluctuating high temperatures,
28 °C for 12 h and 34 °C for 12 h; and (ii) chemical stress, where larvae were
reared on food containing 8.2% ETOH. The biotic stress treatments were:
(i) bacterial stress, where larvae were reared on standard food to which four
drops of the pathogenic bacteria Serratia marcescens were added (~4.8 × 1010
CFU per vial); and (ii) intraspecific competitive stress, where larvae were reared
on food diluted to 33% (1/3 × ) by adding 2/3 agar (18 g l − 1) to 1/3 of the
standard 1 × food medium. The conditions used for each stress treatment were
chosen on the basis of preliminary experiments demonstrating an average
45–50% reduction in the survival of outbred larvae relative to control
conditions. Therefore, the level of stress was standardized as much as possible
across all four stress treatments.
Another form of standardization was the use of a ‘standard’ competitor.
This provided an internal reference for each vial that can control for a common
experimental problem that arises when larval interactions may influence
survival. For example, a group of weak competitors can have the same average
survival as a group of strong competitors, whereas a mixture of the two would
show a substantial survival inequality (Gale, 1964). The use of the standard
competitor allows such large differences to be detected.
To set up the experimental vials, each inbred and outbred bottle population
described above was transferred to an empty glass bottle capped with a petri
dish containing standard food medium and allowed to lay eggs for a period of
8 h. First instar larvae were collected in groups of 100 larvae for up to 8 h from
the laying dish, so that all larvae were within ± 4 h in age. For each
experimental vial, larvae were transferred using a paintbrush to vials containing
10 ml of food medium in the ratio of 100 experimental larvae to 150 of a
standard competitor spa (D.mel laboratory stock with recessive spa eye
mutation). All vials were maintained at 25 °C except for the heat-stressed vials.
Fitness measures
Larval to adult survival was measured under the four stress and the benign
control conditions. Males that survived these treatments were used to measure
mating success.
Larval to adult survival. Following the set up of first instar larva in the
experimental vials (day 1), eclosing adults first appeared on days 9–10 at 25 °C or
on days 8–9 at 33/28 °C. All emerging adults were counted and removed every
3–4 days until approximately day 20–21 at 25 °C and day 19–20 at 33/28 °C, by
which time the number of first generation progeny emerging per vial had
typically diminished to zero over the final 3–4-day counting interval (day ~ 19–21
or ~ 18–20, respectively, for temperature regimes) and a large number of dark
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pupae representing the next generation were observed. There was always
distinction between the first and second generation.
Larval–adult survival was measured in two ways: as the proportion
larvae surviving to eclosion (LS) and as the larval competitive index
which is the proportion that eclosed per vial of the test line relative
proportion of spa competitors (Knight and Roberston, 1957).
a clear
of test
(LCI),
to the
Male mating success. Adult males that emerged from the larval–adult survival
assay were used to measure male mating success using methods described by
Enders and Nunney (2010). In summary, five virgin males were randomly
selected from each vial and held in a fresh vial until they were 5–8 days old,
when they were placed with 15 unrelated virgin competitor spa males and 10
unrelated virgin spa females in new vials containing 10 ml food for 2 h at 25 °C.
Females were removed using light anesthesia and transferred individually to
new vials. After ~ 2 weeks, the progeny of each spa female were scored for eye
color to determine her mate (100% wild type if mated to a test male, 100% spa
if mated to spa competitor male).
Male mating success was measured both as a proportion of test males mating
(MS) and, relative to the standard competitor spa, as the male competitive
index (MCI), which was defined as the proportion of females inseminated by
test males divided by the proportion of females inseminated by the standard
competitor spa males.
Cumulative male fitness was calculated by multiplying LS (LCI) and male
mating success (MCI) for each of the 100 tests described above under each
experimental treatment (four stresses and control).
Data analyses
All analyses below were performed in SAS Version 9.1 for Windows. In all
analyses of covariance (ANCOVA), interactions between the continuous
variable and all categorical variables were first confirmed to be nonsignificant
(P40.05) before proceeding. In all ANCOVA and analyses of variance
(ANOVA), highly nonsignificant interactions (P40.25) among categorical
variables were excluded from the final model.
We calculated the stress level (S_LEVEL) according to Fox and Reed (2011)
using the absolute LS of the outbred lines under benign control conditions
(LSbenign) relative to their survival under each of the four stress treatments
(LSstress);
SLEVEL ¼ 12 LSstress =LSbenign
ð1Þ
where, within each experimental block of each source population, LSstress was
estimated as the mean of the five test values corresponding to the average of
three replicates per outbred line for each of the four stress types, and LSbenign
likewise for the control conditions, giving 16 total values of S_LEVEL. The
experimental design does not allow for meaningful pairing of individual control
and stress replicates to calculate stress levels; we therefore averaged across
replicates for each outbred line to calculate stress level. By definition, the
control treatment had a stress level equal to zero. To determine whether
S_LEVEL differed across the stress treatments, we used a one-way ANOVA with
STRESS (Heat, Ethanol, Bacteria, Competition), and post hoc multiple
comparisons were made using a Tukey test. LCI was not used to estimate
stress level because it is a relative measure, that is, if both the outbred and
spa larvae responded equally to stress, then the calculated stress level would
always be zero.
We also measured post-exposure effects of a stressful larval rearing
environment using outbred male mating success (MCIoutbred) and cumulative
male fitness (LCI × MCI = CUMoutbred). S_EFFECT was calculated as the
relative reduction in the fitness (MCIoutbred or CUMoutbred) of outbred males
reared under stressful and benign conditions:
SEFFECT ¼ 12 Fitnessstress =Fitnessbenign
ð2Þ
where, within each block of each source population, Fitnessstress was estimated
for each of the four stress types and Fitnessbenign for the control conditions
by averaging across the five test values calculated from five outbred lines, giving
16 total values of S_EFFECT. To determine whether S_EFFECT calculated for
both MCIoutbred and CUMoutbred differed across the stress treatments, we used a
Stress and purging reduce inbreeding depression
LS Enders and L Nunney
307
one-way ANOVA, and post hoc multiple comparisons were made using a Tukey
test adjusted for multiple testing.
ID was analyzed for both LS and male mating success using the raw
percentage of larvae surviving (LS) or males mating (MS), the competitive
index (LCI or MCI), and the multiplicative measure of cumulative male fitness
(LCI × MCI). Overall conclusions did not differ between analyses of raw
percentages (LS or MS) and competitive indices (LCI and MCI), therefore we
only report results using LCI and MCI for analysis of difference in ID.
The competitive indices were log-transformed and used to calculate the number
of lethal equivalents (β), a measure commonly used to compare the effects of
inbreeding on fitness across studies, species/taxa and environments
(Armbruster and Reed, 2005) using:
lnðfitnessÞ ¼ bF þ c
ð3Þ
(Morton et al, 1956), where F is the level of inbreeding (F = 0, 0.25 or 0.50),
fitness was either LCI, MCI or cumulative male fitness, and c is the intercept.
We calculated the number of lethal equivalents for both populations across five
environmental treatments in two replicate blocks of the experiment (20 total
values). This approach assumes that deleterious loci have independent effects
on fitness and predicts ln(fitness) decreases linearly with increased inbreeding.
We tested the linearity of regression (3) by comparing the first and second
order polynomial regressions using an F test to determine whether inclusion of
a quadratic term significantly improved model fit (that is, there was curvature
in the relationship between ID and F level).
Although we attempted to standardize the stress levels imposed
(the reduction in outbred LS under stressed relative to control conditions),
variation was to be expected. To determine the potential effect of variation in
larval stress level on ID, we performed two analyses: (i) ANCOVA on levels of
ID (β) for LCI, MCI and cumulative fitness across the four stressful larval
treatments (STRESS: heat, ethanol, bacteria and competition) with S_LEVEL
calculated using equation (1) as a covariate and (ii) ANCOVA on levels of ID
(β) for LCI, MCI and cumulative fitness across the five experimental treatments
(ENV: control, heat, ethanol, bacteria and competition) using larval S_LEVEL
as a covariate. In this second ANCOVA, β and S_LEVEL were calculated for
each environment (including the control conditions) within each of the 4
block/population combinations (20 total), and stress level was defined by:
SLEVEL ¼ 12 LSENV =Avg LSBenign
ð4Þ
where Avg LSbenign was the average of all control replicates across the 2
blocks × 2 populations. This differed from the calculations based on equation
(1), which defined each stress level relative to its block × population control
(LSbenign), thus defining all control S-LEVEL as zero.
To determine whether the number of lethal equivalents differed under
benign (βbenign) and stressful (βstress) conditions, an ANOVA was run for each
fitness measure (LCI, MCI, cumulative fitness) using the following variables:
ENV (Control, Heat, Ethanol, Bacteria, Competition), POP (Gala,Mayo) and
BLOCK. ENV was a fixed effect, whereas BLOCK and POP were random
variables. The following a priori independent planned comparisons of lethal
equivalents were performed: (i) Control (Benign) vs Stress Conditions;
(ii) Abiotic vs Biotic Stresses; (iii) Ethanol vs Heat; and (iv) Bacteria vs
Competition. Post hoc multiple comparisons were calculated using a Tukey test
with Games and Howell’s correction for unequal variances to determine
whether ID differed across the environmental treatments.
Intragenerational purging of genetic load predicts a negative correlation
between the level of ID (β) for LS and male mating success (MCI). LS is the
relevant variable in this prediction (instead of LCI), because LS is a direct
measure of the potential purging. We calculated the correlation between ID for
LS and ID for male mating success, using each population (Gala and Mayo),
block (Block I and II) and environmental condition as data points. An
ANCOVA was also run to determine whether ID post exposure to stress
(B_MCI: ID measured as lethal equivalents for male mating success) was affected
by ID during the larval period (B_LS: lethal equivalents for larval survival),
category of stress (S_CATEGORY: abiotic vs biotic) and the specific STRESS
nested within S_CATEGORY (that is, Heat vs Ethanol; Bacteria vs Competition).
We also tested for purging effects in the outbred populations by comparing
post-exposure stress effects in males and stress levels during larval exposure. An
ANCOVA was run to determine whether stress effects in males (S_EFFECT)
were affected by stress levels during the larval period (S_LEVEL), category
of stress (S_CATEGORY: abiotic vs biotic) and the specific STRESS nested
within S_CATEGORY.
RESULTS
Stress level: exposure and post-exposure fitness effects on outbred
individuals
Stress level was quantified as the decrease in survival of outbred larvae
subject to stress relative to their survival under benign conditions
(equation (1) using the absolute level of LS). The mean stress level
across all four stress types was 0.42 ± 0.03, that is, there was an average
42% reduction in outbred LS relative to benign conditions. ANOVA
revealed significant variation in the level of larval stress induced by the
four stressors (Figure 1: F3,12 = 5.33, P = 0.01). Stress level was
homogeneous across three of the four stressors (averaging
0.46 ± 0.02 across ethanol, bacteria and larval competition), but
significantly lower for the high temperature treatment (0.29 ± 0.01).
We quantified the post-exposure effects of a stressful larval
environment using the relative competitive mating success (MCI) of
outbred males reared in benign and stressful conditions (equation
(2)). Experiencing stress during larval to adult development reduced
male mating success across all four stressors by an average of 57 ± 3%.
This post-exposure reduction in outbred male fitness relative to
benign conditions (post-exposure effects of stress) varied significantly
across the different stress types (Figure 1: F3,12 = 32.6, Po0.001).
Post hoc testing indicated the biotic stresses (competition and bacteria)
induced equivalent post-exposure loss of fitness (0.57 ± 0.02). Among
the abiotic stressors, heat induced a greater post-exposure reduction in
fitness than all other stressors (0.79 ± 0.01, P = 0.01), whereas ethanol
caused less of an effect post exposure (0.35 ± 0.02, P = 0.01–0.02).
The overall effect of early developmental (larval) stress was
quantified as changes in cumulative outbred male fitness, a measure
reflective of both direct and residual effects of larval stress. On average,
cumulative outbred male fitness was reduced by 50 ± 3% across
all stressors relative to benign conditions. However, there was a
significant variation in this fitness reduction across stress types
(F3,12 = 6.06, Po0.01), owing to males exposed to bacterial stress
performing significantly worse than those exposed to ethanol
(0.60 ± 0.09 vs 0.43 ± 0.06, Po0.01; Figure 1).
ID during exposure to stress
Stress imposed during the larval period always resulted in greater ID
than under benign conditions, measured as decreased LS in inbred
lines (Table 1a). Assuming that ID is due to a set of independently
acting loci, it can be converted to lethal equivalents using
the regression equation (3). To test this assumption, we examined
the linearity of the regression (Table 1). There were no indications of
nonlinearity when heat, bacteria or competition were the stressors;
however, under control conditions and ethanol stress, there was a
drop-off in the effect of increasing inbreeding from F = 0.25 to 0.5
compared with F = 0 to 0.25 (Po0.001, 0.01, respectively).
We examined the effects of larval rearing environment on levels of
ID by performing an ANOVA on lethal equivalents derived using
the LCI (βLCI), followed by a series of planned comparisons within the
ANOVA that partitioned the effect of each type of stress into
hypothesis-testing components (Figure 2a; Table 2a). The planned
comparison of βLCI in control vs the four stress conditions demonstrated that the overall effect of stress was significant (Po0.01).
Under the four stressful conditions, the average number of lethal
equivalents expressed for LS was threefold higher (βLCI = 1.47 ± 0.18)
relative to the benign control conditions (βLCI = 0.46 ± 0.01)
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Figure 1 The level of stress measured as a reduction in the fitness of outbred individuals relative to control flies. Direct effects of stress were measured using
LS resulting from exposure to each stressor (heat, ethanol, bacteria and larval competition), and the post-exposure effects of stress were measured using the
MCI. The two fitness effects were combined (LCI × MCI) to estimate the cumulative effect of stress on male fitness. Letters indicate significant differences
between stress types for each fitness measure (Po0.05).
Table 1 Summary of the mean (± s.e.) larval survival and male mating success under benign and stressful conditions under three levels
of inbreeding
Inbreeding
a. Larval survival
F = 0 (Outbred)
Control
Heat
ETOH
Bacteria
Competition
LCI
1.64 ± (0.03)
3.85 ± (0.12)
1.44 ± (0.04)
1.20 ± (0.02)
1.78 ± (0.10)
% survival ( LS)
86.58 ± (0.67)
N = 52
62.02 ± (1.41)
N = 53
47.46 ± (1.60)
N = 54
45.73 ± (1.32)
N = 52
47.60 ± (1.65)
N = 42
F = 0.25
LCI
% survival (LS)
1.34 ± (0.02)
75.02 ± (0.92)
2.70 ± (0.08)
49.79 ± (1.05)
1.07 ± (0.04)
37.10 ± (1.63)
0.90 ± (0.34)
34.58 ± (1.50)
1.17 ± (0.07)
32.17 ± (1.51)
F = 0.50
LCI
N = 92
1.29 ± (0.02)
N = 94
1.86 ± (0.08)
N = 96
1.00 ± (0.04)
N = 88
0.70 ± (0.04)
N = 59
0.72 ± (0.05)
% survival (LS)
72.70 ± (0.97)
N = 85
37.16 ± (1.38)
N = 90
36.41 ± (1.74)
N = 92
28.41 ± (1.70)
N = 81
22.12 ± (1.44)
N = 60
LCI
% survival (LS)
0.46 ± (0.01)
0.34 ± (0.05)
1.65 ± (0.32)
1.14 ± (0.29)
0.92 ± (0.15)
0.62 ± (0.22)
1.19 ± (0.36)
1.16 ± (0.38)
2.12 ± (0.18)
1.80 ± (0.31)
F1,9 (P value)
23.19 (o 0.001)
0.03 (NS)
22.53 (o 0.01)
0.67 (NS)
0.32 (NS)
β
Curvature
b. Male mating success
F=0
MCI
3.17 ± (0.25)
0.67 ± (0.04)
2.04 ± (0.20)
1.34 ± (0.13)
1.35 ± (0.09)
% Matings
71.45 ± (0.02)
N = 52
37.93 ± (0.02)
N = 54
60.1 ± (0.02)
N = 53
51.86 ± (0.02)
N = 53
54.56 ± (0.02)
N = 42
F = 0.25
MCI
% Matings
1.21 ± (0.09)
50.74 ± (0.01)
0.41 ± (0.03)
26.79 ± (0.01)
1.07 ± (0.08)
46.92 ± (0.02)
0.93 ± (0.07)
43.85 ± (0.02)
0.94 ± (0.06)
45.60 ± (0.02)
F = 0.50
MCI
N = 84
0.97 ± (0.08)
N = 86
0.31 ± (0.03)
N = 82
0.88 ± (0.08)
N = 75
0.82 ± (0.10)
N = 59
0.67 ± (0.05)
% Matings
45.09 ± (0.02)
N = 80
21.36 ± (0.01)
N = 77
42.05 ± (0.02)
N = 70
39.94 ± (0.02)
N = 57
36.90 ± (0.02)
N = 48
MCI
% matings (MS)
2.32 ± (0.23)
4.20 ± (0.03)
1.70 ± (0.28)
3.55 ± (0.07)
1.67 ± (0.37)
4.04 ± (0.05)
1.11 ± (0.15)
3.90 ± (0.05)
1.51 ± (0.14)
4.00 ± (0.01)
F1,9 (P value)
11.39 (o 0.001)
0.39 (NS)
3.06 (NS)
1.43 (NS)
o0.01 (NS)
(Outbred)
β
Curvature
Abbreviations: LCI, larval competitive index; LS, larval survival; MCI, male competitive index; MS, male mating success; NS, not significant. (a) Larval survival was measured both relative to the
standard competitor (LCI) and in absolute terms (LS) as the percent survival in five environments: control, two abiotic stresses (heat, ethanol) and two biotic stresses (pathogenic bacteria, increased
competition), (b) Male mating success was measured post stress in the same manner as larval survival (MCI and MS). The results (based on N replicate vials) were pooled across the two original
stock populations (Gala and Mayo), because neither block nor source population had a significant effect (see Table 2). Curvature measured whether the decline in fitness (ln(LCI)) decreased with
increasing F level relative to regression equation (3) linking ID to lethal equivalents (β).
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Stress and purging reduce inbreeding depression
LS Enders and L Nunney
309
Table 2 ANOVA comparing inbreeding depression under benign and
stressful conditions for (a) larval survival (LCI), (b) male mating
success (MCI) and (c) cumulative male fitnessa
Source
DF
MS
F
P value
ENV
Stress vs Control
4
1
1.644
6.09
10.97
0.005
0.005
Biotic vs Abiotic
Ethanol vs Heat
1
1
1.84
3.51
0.195
0.080
5.74
2.88
0.030
0.114
0.70
0.420
a. Larval survival
Larval Competition vs Bacteria
POP
BLOCK
Error
b. Male mating success
ENV
1
1
0.776
1
13
0.188
0.270
4
0.755
5.43
0.017
Stress vs Control
Biotic vs Abiotic
1
1
7.45
1.74
0.016
0.207
Ethanol vs Heat
Larval Competition vs Bacteria
1
1
0.01
1.38
0.940
0.258
0.02
1.72
0.902
0.222
POP
BLOCK
1
1
0.003
0.573
13
0.139
c. Cumulative male fitness
ENV
4
0.947
2.04
0.147
Stress vs Control
Biotic vs Abiotic
1
1
0.19
0.29
0.688
0.599
Ethanol vs Heat
Larval Competition vs Bacteria
1
1
2.31
6.04
0.149
0.030
0.64
0.24
0.439
0.632
Error
POP
BLOCK
Error
1
1
0.295
0.112
13
0.436
Abbreviations: ANOVA, analysis of variance; DF, degrees of freedom; LCI, larval competitive
index; MCI, male competitive index; MS, male mating success. Two populations of
D. melanogaster (POP: Gala and Mayo) were used and the experiment was replicated twice
(BLOCK). Interactions were nonsignificant (P40.25) and removed from the models. Bold values
indicate Po0.05.
aThe analysis compares inbreeding depression (lethal equivalents) under control conditions and
the four stress treatments (ENV), and includes four independent planned comparisons.
Figure 2 ID for LS and male mating success under benign and stressful
conditions. The number of haploid lethal equivalents (β ± s.e.) was measured
relative to a standard competitor using (a) the LCI, (b) the MCI and
(c) cumulative male fitness (LCI × MCI). The number of lethal equivalents for
the control conditions and each individual stress treatment are shown.
Significant differences in levels of ID (β) were determined using post hoc
planned comparisons, which are represented in a tree (NS P40.05,
*Po0.05, **Po0.01, ***Po0.001).
(Table 1a). Three additional planned comparisons were used to
determine whether the stress type affected the magnitude of ID
(Figure 2a; Table 2a). Overall, abiotic and biotic stresses caused similar
levels of ID. Within the biotic stress category, the two stresses differed
(Po0.05), with larval competition resulting in higher ID. Although
the two abiotic stresses, ethanol and heat, were not significantly
different (P = 0.08), it was notable that heat did result in a higher level
of stress-induced ID even though the level of the heat stress was
significantly lower (see above).
Although we attempted to minimize variation in stress levels
imposed by the different stressors, it was important to determine
whether this variation significantly affected the ID detected. For this
purpose, we performed an ANCOVA comparing βLCI across all larval
stressors (STRESS: heat, ethanol, bacteria, competition) with larval
stress level (S_LEVEL) as a covariate. Initial testing showed the effect
of S_LEVEL on ID did not vary across stressors (STRESS × S_LEVEL,
F1,8 = 2.15, P = 0.17) and we excluded the interaction. The simplified
model showed no significant effect of stress levels (S_LEVEL,
F1,11 = 2.65, P = 0.10), and ID did not vary significantly across these
larval environments, (STRESS, F3,11 = 2.65, P = 0.10). Adding the
control data to the analysis showed a significant effect of S_LEVEL
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Stress and purging reduce inbreeding depression
LS Enders and L Nunney
310
(F1,14 = 6.68, P = 0.02), an effect driven by the inevitable near zero
values in the benign controls. With inclusion of control data, ID varied
significantly across larval environments (F4,14 = 3.48, P = 0.04),
in agreement with the original ANOVA of βLCI showing stress
increased ID relative to control conditions.
ID post exposure to stress
The effect of stress imposed during the larval period always reduced
the ID seen in subsequent male mating success, as measured by MCI
or MS (Table 1b). Tests of the regression equation (3) linking ID to
lethal equivalents revealed linearity of the fit to equation (3) in the
groups previously subjected to stressful conditions (Table 1); however,
the controls showed a drop-off in the accumulation of ID at the higher
level of inbreeding (Po0.001). Planned comparisons within the
ANOVA analysis of ID for male mating (βMCI) showed that this
post-stress reduction was significant (Po0.02) and there were no
additional effects due to differences among the stressors (Table 2b;
Figure 2b). The number of lethal equivalents for competitive
mating success expressed in males exposed to stressful conditions
(βMCI = 1.60 ± 0.14) was on average 30% lower than males reared
under benign conditions (βMCI = 2.32 ± 0.23) (Table 1b). Exposure to
abiotic and biotic stress caused similar levels of ID for male mating
success, as did the two abiotic stressors (heat and ethanol) and two
biotic stressors (bacteria and competition) (Table 2b).
We further analyzed βMCI using an ANCOVA including the larval
stressors (STRESS: heat, ethanol, bacteria, competition) and
S_LEVEL experienced during the larval exposure to these stressors
to determine whether the experimental variation in stress levels
experienced as a larva resulted in variation in the magnitude of
ID expressed as an adult male. Initial testing showed no interaction
between these two variables (STRESS × S_LEVEL, F3,8 = 0.01,
P40.25) and the simplified model showed that the level of ID
for male mating (βMCI) varied with larval S_LEVEL (F1,11 = 5.12,
P = 0.04, slope = 4.12 ± 1.72), but not the specific stressor
(STRESS: F3,11 = 1.35, P = 0.31). Adding the control data increased
the significance of S_LEVEL (F1,14 = 15.10, P = 0.001), but again
there was no effect of environment (ENV: F1,14 = 1.15, P = 0.37),
where ENV adds the control to the four STRESS environments.
Cumulative ID and intragenerational purging
After combining the inbreeding effects apparent at the stressed larval
stage with those affecting later male mating success, ANOVA showed
that exposure to stressful conditions during larval development
did not cause any cumulative stress-induced ID of males (Stress vs
Control, Table 2c; Figure 2c). The number of cumulative lethal
equivalents (BCUM) expressed in males that experienced benign
conditions was 2.92 ± 0.19 vs 2.76 ± 0.20 on average across the four
stresses. The additional planned comparisons showed that there was
no difference in the level of ID resulting from abiotic vs biotic stresses.
However, although there was no significant difference between ID
caused by the two abiotic stresses, the two biotic stresses did differ,
with bacterial infection resulting in significantly lower ID than larval
competition (Table 2c; Figure 2c).
An ANCOVA of cumulative male ID that included larval stress level
(S_LEVEL) showed no interaction between S_LEVEL and ENV four
stresses and control) (F4,10 = 1.37, P = 0.31), and the simplified model
showed no effect of S_LEVEL (F1,14 = 2.88, P = 0.11, slope = 4.50 ± 2.20)
or the five experimental treatments (ENV: F4,14 = 3.00, P = 0.06).
We exploited the experimental variance among the replicates across
the two source populations and two experimental blocks of each of the
four stress treatments to test for evidence of intragenerational purging
Heredity
of genetic load. The prediction is that greater levels of ID during
the stressed period (βLS) should result in lower levels of ID post
exposure for male mating success (βMCI). We used LS rather than
LCI (a measure including competitive success) because LS is a direct
estimate of potential purging. ANCOVA of lethal equivalents for
male mating success showed a significant interaction between
S_CATEGORY (abiotic vs biotic) and βLS (F1,8 = 9.4,P = 0.015). Thus,
the analysis was split by stress category and we found that ID for male
mating (βMCI) was significantly negatively correlated with ID for LS
(βLS) in the biotic stresses (P = 0.04), whereas for the abiotic stresses,
there was a nonsignificant positive slope (Table 3; Figure 3). Although
the two biotic stresses showed the same slope, their elevations
differed (P = 0.02) possibly indicating more effective purging by the
bacterial treatment.
Outbred populations did not show evidence of intragenerational
purging, that is, there was no significant relationship between stress
level of the outbred larvae and stress effect for mating success of
outbred males (Figure 4). ANCOVA of stress effect in male mating
(S_EFFECT) showed no indication of a correlation with stress levels
experienced as larva (S_LEVEL) (slope = − 0.52 ± 0.36, F1,8 = 0.48,
P = 0.51) and this relationship did not vary across the two broad
stress categories (S_LEVEL × S_CATEGORY: F1,8 = 1.93, P = 0.2).
Overall, there were no differences between stress categories
(S_CATEGORY: F1,8 = 2.15, P = 0.18) or across individual stressors
(S_TYPE (S_CATEGORY): F1,8 = 1.39, P = 0.30).
DISCUSSION
The questions addressed in this study were: (i) Does stress consistently
increase ID in the life history stage subject to exposure? (ii) Does any
increase in ID persist beyond the period of stress? (iii) How do these
stress effects combine to affect overall fitness in inbred populations?
and (iv) Do different stressors affect ID in different ways? Our results
demonstrate that exposure to heat, ethanol, pathogenic bacteria and
larval competition causes consistent and significant increases in the
expression of lethal equivalents for larval–adult survival relative to
benign conditions (Figure 2a). However, this increase in ID observed
during larval exposure to stress did not extend to later life history
stages. ID was significantly reduced for males surviving stressful
Table 3 ANCOVA comparing the level of inbreeding depression
(lethal equivalents) for male mating success (MCI) after exposure as
larvae to abiotic or biotic stress
Source
Abiotic stresses
STRESS TYPE
DF
MS
F
P value
1
0.315
1.130
0.348
ID_LS
Slope = 0.50 ±0.40
1
1.081
3.880
0.120
Error
4
0.278
Biotic stresses
STRESS TYPE
1
0.879
10.400
0.023
ID_LS
Slope = -0.71 ± 0.17
1
0.665
7.870
0.038
Error
4
0.278
Abbreviations: ANCOVA, analysis of covariance; DF, degrees of freedom; LS, larval survival; MCI,
male competitive index; MS, male mating success. The analysis included STRESS TYPE: abiotic
stress (heat and ethanol) and biotic stress (bacteria and larval competition), and the level of
inbreeding depression expressed in larval survival during exposure to stress (ID_LS) as a
covariate. The analysis was split by category of stress (abiotic vs biotic) owing to a significant
interaction between stress category and ID_LS (F1,8 = 9.4 P = 0.015). Nonsignificant
interactions with the covariate (ID_LS × STRESS TYPE) were removed and the model was rerun.
Bold values indicate Po0.05.
Stress and purging reduce inbreeding depression
LS Enders and L Nunney
311
Figure 3 Relationship between ID in lethal equivalents expressed under
stress and post stress. ID under stress was estimated using LS (βLS) and
post stress using male mating success (βMCI). For each of the stress
treatments, four points are shown corresponding to the average lethal
equivalents for each replicate (Block I and II) in each population (Gala and
Mayo). The fitted line is indicated for each stress, although the slopes only
differ between biotic and abiotic treatments (Table 3).
Figure 4 Relationship in outbred individuals between stress level during
larval exposure and the post-stress effect after exposure on male mating.
Stress level was measured using LS and post-exposure stress effects using
male mating success (MCI).
larval conditions compared with those that developed in a benign
environment (Figure 2b), indicative of purging of deleterious alleles
during exposure to stress. When examined cumulatively in males,
we found the paradoxical result that in spite of the large direct effect of
stress in increasing larval ID, the net ID was no different under
stressful and benign conditions (Figure 2c), suggesting that because of
intragenerational stress-induced purging, stress had minimal
population-level effects on the overall expression of genetic load.
Is stress type an important determinant of ID?
The central role of stress level in determining ID raises the question of
the extent to which the type of stress matters. Such comparisons of
different stressors require that stress level be standardized (Fox and
Reed, 2011) or, if not, differences should be accounted for in the
analysis. We followed this protocol (with an average stress level of 42%)
and found no differences in ID when larvae were directly exposed to
stress based on the biotic vs abiotic grouping (Table 2), although within
these two groups, there were marginally significant differences in the
level of ID (competition4bacteria; heat4ethanol; see Figure 2a).
After the stress had been removed, previously stressed male flies had
significantly lower ID compared with unstressed control flies, an effect
that also did not vary by stress type (Tables 1b and 2b; Figure 2b).
These results are in line with previous work in Drosophila using a
similar level of larval competitive stress (stress level = 52%), which
found higher ID during exposure (larval–adult survival) but not for
post-stress adult reproductive fitness traits (Enders and Nunney, 2010).
We also examined whether different stressors resulted in an
accelerating or decelerating expression of ID as inbreeding increased
from F = 0.25 to F = 0.5 relative to expectation (see equation (3)).
The ID resulting from the stressors fitted closely to expectation,
consistent with deleterious alleles acting independently to reduce
fitness. The only exception was the effect of ethanol stress on larval
ID. In contrast, we found a highly significant decelerating effect under
control conditions both for larval and adult male ID. Thus, there is no
evidence that deleterious alleles are acting synergistically under
stressful conditions to decrease fitness (that is, there is no negative
epistasis), and that under non-stressful control conditions, there is
positive epistasis, so that combinations of deleterious alleles are less
damaging than would be expected.
Overall, stress type was found to cause only minor differences in
both the increase in ID expressed during direct exposure and the
decrease in ID observed in post-exposure male mating success
(Figures 2a and b). Thus, our results do not support the findings of
Yun and Agrawal (2014), who found that ID was strongest when
competition was intense. We found that, although competition
resulted in a slightly higher ID than the other stressors, its
effect was very similar to that of heat, while the other two stressors
(ethanol and bacteria) induced very similar levels of ID (see Figure 2a),
pairings that suggest no particular pattern. The distinction between
biotic and abiotic stressors has been found to be important in the
study of plant responses to stress, where different signaling pathways
can be involved (see review by Atkinson and Urwin 2012). This is
notably the case when biotic and abiotic stressors interact; however, in
our experiments, we have not investigated the possibility of such
interactions. Although we found no differences in the magnitude of
the effect of these two types of stressors, there were indications in the
data that abiotic stressors may cause additional damage (see below).
Similarity in the physiological and cellular damage induced by
different stressors, as well as overlap in molecular level defenses, could
contribute to minimizing differences in the expression of ID across
stress types. Although different stressors can have variable effects,
similar changes in gene expression and protein abundance have also
been observed under a variety of stressors in plants and animals
(Kültz, 2005; Storz and Hengge, 2011). For example, work in
Drosophila has shown that common molecular responses are present
in populations selected for tolerance to a variety of abiotic stressors
such as heat, starvation and desiccation (Sørensen et al., 2007).
In addition, inbreeding alters the expression of genes involved in
primary metabolic processes and general stress defense (Kristensen
et al., 2005; Kristensen et al., 2006), which could render individuals
generally more vulnerable to a variety of stressors. If molecular
mechanisms underlying defense responses were predominately shared
among the stress types used in the current study, this would also
minimize variation in the expression of ID.
Despite our efforts to equalize the level of stress during larval
development across all four types of stress, heat treatment was found
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Stress and purging reduce inbreeding depression
LS Enders and L Nunney
312
to be less stressful than the other three stresses (Figure 1). When this
variation was accounted for using ANCOVA, the relationship between
stress level and ID was not significant for LS or cumulative fitness,
indicating that lower mortality under heat stress was not affecting the
observed patterns in ID.
Stress-induced intragenerational purging vs physiological weakening
Stress is commonly viewed as a cumulative phenomenon, whereby
exposure early in life causes an overall physiological weakening that is
predicted to amplify ID across multiple life history stages even after
the source of stress is removed (Hoffmann and Parsons, 1991;
Kristensen et al., 2003; Armbruster and Reed, 2005; Waller et al.,
2008). Contrary to this view, our results demonstrated that early
exposure to stress consistently reduced ID in later life history stages
(Figure 2b). Intragenerational purging appears to be the explanation:
exposure to larval stress purges less fit larvae that in turn causes a
reduction in genetic load in surviving males, resulting in enhanced
performance relative to unstressed individuals.
Intragenerational purging depends on genetic variation within an
inbred population and is predicted to occur if early and late fitness
components are correlated owing to some genes having pleiotropic
effects important for both (Whitlock and Bourguet, 2000; Whitlock
and Agrawal, 2009). Maintaining metabolic efficiency under stressful
conditions has been hypothesized to be important for energetically
costly processes such growth, development and mating success
involving courtship and competitive interactions, which predicts
stress-induced associations between diverse fitness components
(Parsons, 1997; Parsons, 2007). For example, the insulin signaling
pathway regulates growth and development in both larva and adult
Drosophila (see review in Garofalo, 2002). Thus, recessive deleterious
mutations in these pathways could reduce survival and body size, and
could therefore adversely affect both LS and male mating success.
Such correlations between fitness components are necessary
for intragenerational purging, and predict a negative relationship
between ID during the larval period (which measures the opportunity
for purging) and ID for male mating success (which measures the
consequences of purging). We first eliminated the possibility that such
a correlation could arise from a direct relationship between the traits
unrelated to purging by showing that in outbred flies, there was no
significant negative correlation between larval stress and post-stress
mating success relative to the unstressed control (Figure 4). Next,
we established that, although abiotic and biotic stressors induced
equivalent levels of enhanced larval ID and reduced male mating ID,
there were significant differences in the predicted correlation (Table 3;
Figure 3). Biotic stressors showed a significant negative relationship
consistent with intragenerational purging. Abiotic stressors showed a
nonsignificant positive relationship between ID during and after
exposure (Figure 3), suggesting that, although purging appeared to
be important (causing the reduced ID for male mating success;
see Figure 2b), something else was also affecting the outcome. We
propose that this pattern seen with the two abiotic stresses could arise
from a combination of purging and a general stress-induced physiological weakening. Extensive work in a variety of animals shows longterm protein damage and impaired cellular functioning can result
from heat and ethanol stress (Hoffmann and Parsons, 1991; Sorensen
et al., 2003; Montooth et al., 2006). Studies in Drosophila found
exposure to high heat and sublethal concentrations of ethanol alters
membrane lipid composition and energy reserves (Sorensen et al.,
2003; Montooth et al., 2006), which could have enduring negative
effects on later life history traits such as female fecundity and male
fertility (Krebs and Loeschcke, 1994; Bokor and Pecsenye, 2000).
Heredity
Both heat and ethanol stress also induce upregulation of heat shock
proteins in Drosophila (Sorensen et al., 2005; Kong et al., 2010),
however, this increased expression can also be costly for growth,
development and longevity (see reviews in Sorensen et al., 2003).
Therefore, irreversible damage to basic cellular functioning and
long-term costs associated with mounting molecular stress defenses
could offset in part the fitness benefits associated with stress-induced
purging of genetic load under abiotic heat and ethanol stress.
Overall, our demonstration that early-life exposure to different
stressors caused increased post-stress fitness is unique, as is our finding
of a potentially important difference between biotic and abiotic
stressors, with abiotic stressors perhaps inducing a persistent physiological weakening at the stress levels that we examined. It will be
interesting to see whether this biotic/abiotic distinction persists in
future studies across a range of stress levels. If the stress-induced loss
of fitness accelerates relative to the fitness gain via purging as the level
of stress increases, then purging is predicted to dominate at low stress
levels, but at higher levels, any further benefits of purging would be
negated by the increasing stress-induced loss of fitness via irreversible
cellular damage. In addition, the purging effect is predicted to decline
with increased inbreeding (because purging relies on the presence of
genetic variation) but the weakening effect works equally well in a
genetically uniform population.
Cumulative patterns of ID
Combining LS and mating success, it was found that cumulative ID
expressed in males was independent of the presence or absence of
stress (Table 2c; Figure 2c). The only indication of any effect of stress
type on this result was a significant difference between the two biotic
stresses, driven mainly by the low cumulative inbreeding in the
bacterial treatment. A similar equalization of the cumulative ID was
apparent in the study of Montalvo (1994), where early development of
perennial Aquilegia caerulea occurred either in the greenhouse or field.
However, unlike previous studies where plants were maintained in
stressful field conditions following early development (Montalvo,
1994; Goodrich et al., 2013), the most plausible explanation for
patterns of cumulative ID observed in the current study is intragenerational purging. We maintained males in a benign environment
following developmental stress, thus avoiding the possibility that
continued exposure to stressful conditions in later life stages and
additional selective pressure could explain the equivalent cumulative
ID observed across environments.
Given a correlation of deleterious effects acting during stressful
and post-stress life history stages, we can expect some degree of
intragenerational purging. However, the magnitude of the effect is
hard to predict, because it depends upon the sensitivity of the different
fitness components to the presence/absence of deleterious alleles under
the conditions being tested. Thus, although it is clear that stress
increases such sensitivity (resulting in increased ID), the sexual
selection acting on male mating success may, depending upon
conditions, result in even greater fitness discrimination. This would
lead to the paradoxical result of lower cumulative ID in stressed
populations. In our experiments, it appears that the stress-related
fitness effects and subsequent mating effects were approximately
balanced, leading to the observed result of equal ID in control and
stressed groups; although it is interesting to note that the cumulative
ID observed following stress with a bacterial pathogen was substantially lower than that observed in the control group (Figure 1c).
Our finding of no net increase in ID with stress when fitness is
evaluated cumulatively brings into question the generality of the
suggestion that increased stress can increase the efficiency of purging
Stress and purging reduce inbreeding depression
LS Enders and L Nunney
313
deleterious alleles at the population level. This has been proposed as a
mechanism useful for alleviating ID and increasing the long-term
fitness of threatened species (Leberg and Firmin, 2008; de Cara et al.,
2013). In particular, we found despite a substantial direct effect of
stress, increasing ID from 0.46 to 1.47 lethal equivalents, the mating
success of males surviving larval stress completely eliminated this
difference. Under such circumstances, stress-induced purging is
unlikely to have beneficial population-level effects.
DATA ARCHIVING
Data available from the Dryad Digital Repository: http://dx.doi.org/
10.5061/dryad.t8344.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
We would like to thank Magdalene Moy for help with Drosophila rearing and
conducting this experiment. This research was supported in part by an NSF
DDIG awarded to L Enders (award no. 0808416).
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